MYC2 Differentially Modulates Diverse Jasmonate …myc2/jin1 (jin1-9) (Anderson et al., 2004) plants...

MYC2 Differentially Modulates Diverse Jasmonate-DependentFunctions in Arabidopsis W

Bruno Dombrecht,a,1 Gang Ping Xue,a Susan J. Sprague,b John A. Kirkegaard,b John J. Ross,c James B. Reid,c

Gary P. Fitt,d Nasser Sewelam,a,e Peer M. Schenk,e John M. Manners,a and Kemal Kazana,2

a Commonwealth Scientific and Industrial Research Organization Plant Industry, Queensland Bioscience Precinct,

St. Lucia, Queensland, 4067, Australiab Commonwealth Scientific and Industrial Research Organization Plant Industry, Canberra, Australian Capital Territory,

2601, Australiac School of Plant Science, University of Tasmania, Hobart, Tasmania, 7001, Australiad Commonwealth Scientific and Industrial Research Organization Entomology, Long Pocket Laboratories, Indooroopilly,

Queensland, 4068, Australiae School of Integrative Biology, University of Queensland, St. Lucia, Queensland, 4072, Australia

The Arabidopsis thaliana basic helix-loop-helix Leu zipper transcription factor (TF) MYC2/JIN1 differentially regulates

jasmonate (JA)-responsive pathogen defense (e.g., PDF1.2) and wound response (e.g., VSP) genes. In this study, genome-

wide transcriptional profiling of wild type and mutant myc2/jin1 plants followed by functional analyses has revealed new

roles for MYC2 in the modulation of diverse JA functions. We found that MYC2 negatively regulates Trp and Trp-derived

secondary metabolism such as indole glucosinolate biosynthesis during JA signaling. Furthermore, MYC2 positively reg-

ulates JA-mediated resistance to insect pests, such as Helicoverpa armigera, and tolerance to oxidative stress, possibly via

enhanced ascorbate redox cycling and flavonoid biosynthesis. Analyses of MYC2 cis binding elements and expression of

MYC2-regulated genes in T-DNA insertion lines of a subset of MYC2–regulated TFs suggested that MYC2 might modulate

JA responses via differential regulation of an intermediate spectrum of TFs with activating or repressing roles in JA sig-

naling. MYC2 also negatively regulates its own expression, and this may be one of the mechanisms used in fine-tuning JA

signaling. Overall, these results provide new insights into the function of MYC2 and the transcriptional coordination of the

JA signaling pathway.

INTRODUCTION

In response to exogenous and endogenous cues, plants synthe-

size various fatty acid derivatives that act as signaling molecules.

Among these, jasmonic acid and its volatile methyl ester, methyl

jasmonate (MeJA), collectively known as jasmonates (JAs), are

the best characterized fatty acid–derived cyclopentanone sig-

nals. JAs modulate a number of vital physiological processes,

including defense against pathogens and insects, wound re-

sponses, secondary metabolite biosynthesis, and flower devel-

opment and fertility (reviewed in Cheong and Choi, 2003).

Receptors for JAs have not been identified, but following the

perception of JA, a number of cellular signaling processes occur

that presumably result in the posttranslational modification (e.g.,

phosphorylation) of upstream regulatory proteins (Rojo et al.,

1998), transcriptional activation of JA-responsive transcription

factors (TFs), and downstream response genes. To date, forward

genetic approaches have been instrumental in the identification

of various genes involved in the JA signaling pathway (Berger,

2002). One of the first JA signaling mediators identified after

map-based cloning of the mutated locus in the coi1 mutant is the

CORONATINE-INSENSITIVE1 (COI1) gene, which encodes an

F-box protein involved in the ubiquitin-proteasome pathway (Xie

et al., 1998). Most JA-regulated responses, including fertility and

defense against pests and pathogens, are altered in the coi1

mutant, suggesting that COI1 acts relatively upstream in the

JA signaling pathway (reviewed in Lorenzo and Solano, 2005).

However, currently, very little is known about negative regulators

of JA-responsive gene expression that might be ubiquitinated

in a COI1-dependent manner. Histone deacetylases (HDACs),

acting as transcriptional repressors of gene expression, have

been implicated as potential COI1 targets. Indeed, COI1 inter-

acts in planta with HDA6, which encodes a histone deacetylase

in Arabidopsis thaliana (Devoto et al., 2002). Another HDAC

involved in JA signaling is HDA19, which functions as a negative

regulator of defense genes positively regulated by ETHYLENE

RESPONSE FACTOR1 (ERF1) (Zhou et al., 2005), an important

TF in JA- and ethylene (ET)-dependent signaling for pathogen

defense (Lorenzo et al., 2003).

1 Current address: Ablynx, Technologiepark 4, 9052 Ghent, Belgium.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Kemal Kazan([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.106.048017

The Plant Cell, Vol. 19: 2225–2245, July 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

MYC2, a basic helix-loop-helix (bHLH) domain–containing TF,

acts as both activator and repressor of distinct JA-responsive

gene expression in Arabidopsis (Lorenzo et al., 2004). MYC2 is

allelic to the JAI1/JIN1 (for JASMONATE-INSENSITIVE1) locus,

which was first identified in a mutant screen for reduced sensi-

tivity of its roots to exogenous JA (Berger et al., 1996). MYC2 is

also known as RD22BP1, RAP-1, or ZBF1 (Abe et al., 1997; de

Pater et al., 1997; Yadav et al., 2005). Despite the potential

significance of MYC2 as a major player in the JA signaling

pathway (Lorenzo and Solano, 2005), only a few MYC2-regulated

genes have been identified to date. These genes include the

JA-responsive pathogen defense genes PDF1.2, CHIB/PR3, and

HEL/PR4 and are negatively regulated by MYC2 (Anderson et al.,

2004; Lorenzo et al., 2004). Consequently, myc2/jin1 mutant

plants show increased resistance to fungal pathogens such as

Plectosphaerella cucumerina, Botrytis cinerea, and Fusarium

oxysporum (Anderson et al., 2004; Lorenzo et al., 2004) and the

bacterial pathogen Pseudomonas syringae (Nickstadt et al.,

2004; Laurie-Berry et al., 2006). In addition, MYC2 positively

regulates the JA- and wound/insect-responsive genes VSP,

LOX, and TAT (Boter et al., 2004; Lorenzo et al., 2004). However,

it is currently unknown whether insect tolerance is compromised

in myc2/jin1. Also unknown is whether MYC2 has additional roles

in modulating other JA-regulated genes and plant functions.

The JA signaling pathway interacts extensively with other

hormonal and developmental signaling pathways, and emerging

evidence suggests that MYC2 plays a pivotal role in modulating

some of these interactions. For instance, MYC2 also acts as a

positive regulator of abscisic acid–dependent drought responses

(Abe et al., 2003) and is required for the suppression of salicylic

acid–dependent defenses during infection by P. syringae (Laurie-

Berry et al., 2006). Interactions between JA and ET as well as JA

and auxin signaling are also known (reviewed in Woodward and

Bartel, 2005), but it is not known whether MYC2 has a role in

regulating such interactions.

Here, we address the following two questions. (1) What other

JA-dependent cellular and phenotypic responses, outside of

disease resistance and wound response, are regulated by

MYC2? (2) How does MYC2 modulate diverse JA responses at

the transcriptional level? Using genome-wide gene expression

analysis of MeJA-treated wild-type and myc2/jin1 plants, we

identified a large number of JA-responsive and MYC2-regulated

genes, including a number of TF genes. In addition, comparative

phenotypic and biochemical analyses of myc2/jin1 wild-type

and myc2/jin1 plants constitutively expressing MYC2 provided

functional evidence that MYC2 positively regulates oxidative

stress tolerance, flavonoid biosynthesis, and insect herbivory

resistance and negatively regulates Trp metabolism, leading to

the JA-dependent synthesis of defensive compounds such as

indole glucosinolates (IGs). Furthermore, we show that JA acti-

vates auxin biosynthesis and that MYC2 is required for the

inhibition of root elongation by auxin transport inhibitors. Finally,

differential expression of diverse TF genes during JA signaling in

the myc2/jin1 mutant along with DNA binding and expression

studies of T-DNA lines of MYC2-modulated TFs have led to the

proposal that MYC2 probably acts through the transcriptional

orchestration of other TFs, which in turn regulate downstream JA

response genes involved in diverse JA-dependent plant processes.

RESULTS

MYC2 Modulates Gene Expression in a

JA-Dependent Manner

A genome-wide transcript analysis was undertaken to identify the

Arabidopsis genes that are regulated by MYC2. In three indepen-

dent biological experiments, wild-type (Columbia [Col-0]) and

myc2/jin1 (jin1-9) (Anderson et al., 2004) plants were either treated

with 0.1 mM MeJA for 6 h or mock-treated as a control. Whole-

genome gene expression of the samples was analyzed using

Affymetrix ATH1 GeneChips (for full experimental details, see

Methods and Supplemental Methods online). Stringent statistical

analysis of the data was performed by means of two-way ANOVA

for the factors of genotype (Col-0 versus jin1-9) and JA treatment

(mock versus 0.1 mM MeJA), and the results are summarized in

Figure 1A. A complete list of genes that are significantly affected

in their expression by either of these factors is also provided in

Supplemental Table 1 online. The Venn diagram given in Figure 1A

summarizes the ANOVA analysis. Most of the MYC2-modulated

genes were also induced by MeJA in the wild-type background

(Figure 1C). In addition, a substantial number of genes (Figure 1A)

had significance (P < 0.05) for interaction between the genotype

and treatment factors (see Supplemental Table 1 online). Overall,

the data from our GeneChip experiments suggest that MYC2

probably modulates the expression of a significant portion of all

Arabidopsis genes. Importantly, comparison of differentially ex-

pressed genes in jin1-9 with their MeJA-responsive expression

in the wild type revealed that the MYC2 dependence of gene

expression was predominantly present under MeJA treatment

(Figures 1B and 1C).

To confirm the outcome of the microarray analysis, three

additional independent biologically replicated time course ex-

periments were set up with Col-0 and jin1-9 with or without MeJA

treatment, and samples were harvested at 1, 3, 6, and 24 h after

treatment. The expression from selected MYC2-regulated genes

was determined by quantitative real-time PCR (Q-RT-PCR),

and the results for genes discussed in some detail are shown

in Figures 3A, 5A, and 6A below and in Supplemental Figure

1 online. For the majority of the tested genes, the Q-RT-PCR

experiments confirmed the microarray results, with significantly

altered expression in at least one time point.

The differentially expressed genes in myc2/jin1 included

known JA- and MYC2-regulated genes (e.g., PDF1.2, CHI/PR3,

HEL/PR4, and VSP) as well as genes involved in a variety of other

JA-regulated functions, such as Trp metabolism, phenylpropa-

noid and flavonoid metabolism, sulfur metabolism, oxidative

stress tolerance, hormone biosynthesis, insect pest resistance,

and senescence (see Supplemental Table 1 online). These results

indicated that MYC2 regulates a wider array of JA responses than

was previously known. In subsequent experiments (see below),

we further investigated phenotypic responses providing a func-

tional role for MYC2 in some of these JA-mediated processes.

MYC2 Negatively Regulates Trp Metabolism

during JA Signaling

The microarray and Q-RT-PCR analyses showed that several

genes involved in Trp biosynthesis and Trp-derived secondary

2226 The Plant Cell

metabolism were differentially expressed in response to MeJA in

the jin1-9 mutant compared with their expression in similarly

treated wild-type plants (Figures 2A and 3A; see Supplemental

Table 1 online). Trp biosynthesis genes (ASB, IGPS, TSA1, and

TSB2), IG biosynthesis genes and their transcriptional regulators

(MYB51, ATR4/CYP83B1/SUR1, APS3, APR3, and ST5a) and

the camalexin biosynthesis gene PAD3/CYP71B15 were con-

sistently expressed at higher levels in jin1-9 than in the wild type

following MeJA treatment, suggesting that MYC2 acts as a neg-

ative regulator of the Trp metabolic pathway during JA signaling

(Figure 2A; see Supplemental Table 1 online).

To determine whether the increased MeJA responsiveness

of the Trp metabolism genes leads to alterations in the activity of

the Trp metabolic pathway in the myc2/jin1 mutant, a 5-methyl-

DL-Trp (5MT) root growth inhibition assay was set up using the

Figure 1. Summary of the Affymetrix GeneChip Experiment.

(A) Statistical analysis of the effect on gene expression of the factors

genotype (Col-0 versus jin1-9) and treatment (mock versus 0.1 mM

MeJA) by two-way ANOVA of the microarray expression data. The

number of genes showing a significant change at P < 0.01 and P < 0.05

(in parentheses) is shown.

(B) and (C) Biplots of the ratios of expression values from the GeneChip

experiments. Genes that are significant for genotype (P < 0.05) are shown

as white diamonds (778 genes). Each data point is the ratio of the averages

of three independent biological replicates. The y axes show the ratio of

average expression levels of MeJA-treated wild-type plants (Col-0) over

mock-treated wild-type plants. The x axes show the ratio of average

expression levels of MeJA-treated myc2 mutant (jin1-9) plants over MeJA-

treated wild-type plants (B) and the ratio of average expression levels of

mock-treated myc2 mutant plants over mock-treated wild-type plants (C).

Figure 2. Schematic Summary of MYC2-Regulated Trp and Flavonoid

Metabolism Genes.

MYC2-regulated Trp and Trp-derived secondary metabolism (A) and

phenylpropoanoid and flavonoid metabolism (B) genes. Significant up-

regulation and downregulation in the MeJA-treated myc2/jin1 mutant

relative to the similarly treated wild-type plants are indicated with up

arrows and down arrows, respectively. Enzymes are depicted in rectan-

gular boxes, and TFs are shown in elliptical boxes. The double arrows

used between the substrates indicate multiple biochemical steps. See

Supplemental Table 1 online for details of the genes.

MYC2 and Coordination of JA Signaling 2227

Figure 3. MYC2 Negatively Regulates Trp Metabolism in a JA-Dependent Manner.

(A) Q-RT-PCR expression analysis of Trp metabolism genes after treatment with 0.1 mM MeJA (black symbols) or mock treatment (white symbols) in

Col-0 (squares) and jin1-9 (triangles). Data are expressed as relative RNA levels ([mRNA]gene/[mRNA]actin) and are means of three biological replicates

(>30 pooled plants each); error bars denote SE.

(B) Root lengths of MeJA-treated (0.5 mM) and 5MT-treated 7-d-old Arabidopsis seedlings. Values (representative of two independent experiments) are

means of >20 seedlings for each treatment/genotype combination; error bars denote SE. Values annotated with different letters are significantly different

(P < 0.01; Tukey’s least significant difference [LSD]). Note that at the relatively low MeJA concentrations used, the difference in root length inhibition

between the wild type and jin1-9 is not significant.

(C) Soluble Trp levels of 5-week-old Arabidopsis plants treated with 0.1 mM MeJA for 24 h. Values are means of three biological replicates (>20 pooled

plants each); errors bars denote SE.

(D) IG levels of 5-week-old Arabidopsis plants treated with 50 mM MeJA for 48 h. I3M, indolyl-3-methyl glucosinolate; 4MI3M, 4-methoxy-indolyl-

3-methyl glucosinolate. Values are means of three biological replicates (>20 pooled plants each); errors bars denote SE. Values annotated with different

letters are significantly different (P < 0.01; Tukey’s LSD).

(E) Free indole acetic acid (IAA) levels of 5-week-old Arabidopsis plants treated with 50 mM MeJA for 48 h. Values are means of three biological

replicates (>20 pooled plants each); errors bars denote SE.

jin1-9 and jin1-10 mutants (Anderson et al., 2004) as well as the

atr2D mutant (Smolen et al., 2002) as a control. The toxic Trp

analog 5MT acts by triggering feedback inhibition of anthranilate

synthase activity without substituting for the nutritional role of Trp

(Bender and Fink, 1998) (Figure 2A). At least two classes of

Arabidopsis mutants show 5MT resistance: mutants with feed-

back resistance mutations in the anthranilate synthase catalytic

subunits and mutants with increased expression of Trp metab-

olism genes (Smolen et al., 2002). Our experiments revealed that

5MT was toxic to the wild type and the two myc2/jin1 mutant

lines at both 25 and 50 mM concentrations. Consistent with

previous observations (Smolen et al., 2002), the control 5MT-

resistant mutant atr2D was virtually unaffected by 25 mM 5MT

(Figure 3B). Importantly, these assays showed that the 5MT tox-

icity leading to the inhibition of root elongation was reduced in the

seedlings germinated in the presence of MeJA, suggesting that

MeJA-mediated alterations in Trp metabolism genes can indeed

lead to changes in the Trp pathway. We found that at 50 mM 5MT,

MeJA treatment significantly enhanced resistance to 5MT in the

myc2/jin1 lines but not in the wild type (Figure 3B). MeJA-

mediated 5MT resistance observed in atr2D at 50 mM 5MT was

similar to that in myc2/jin1 plants. These results indicate that the

MeJA-mediated changes observed in the expression of Trp

pathway genes have altered Trp metabolism to a greater degree

in the myc2/jin1 mutant than in the wild type, leading to a JA-

dependent increase in 5MT resistance.

Interestingly, only the 5MT-resistant mutants with anthranilate

synthase feedback resistance show increased soluble Trp levels,

while mutants (e.g., atr2D) with increased expression of Trp

metabolism genes show reduced Trp levels (Smolen et al., 2002).

We thus measured soluble Trp levels in jin1-9, jin1-10, and wild-

type plants after MeJA treatment. MeJA treatment resulted in

reduced soluble Trp levels in all three lines (P < 0.01, two-way

ANOVA) (Figure 3C). In addition, we found reduced soluble Trp

levels in the myc2/jin1 mutants relative to the wild type under

mock conditions (Figure 3C). Together, these results are sug-

gestive of an increased flux in the Trp pathway following MeJA

treatment and in myc2/jin1 mutants.

MYC2 Is a Negative Regulator of JA-Dependent

IG Biosynthesis

One possible outcome of the increased activity of the Trp

metabolic pathway would be on indole classes of secondary

metabolites and the auxin hormone IAA synthesized through this

pathway. Indeed, Trp is first converted into indole-3-acetaldoxime

(IAOx) by the action of the cytochrome P450 enzymes CYP79B2

and CYP79B3. IAOx is then used in the biosynthesis of IGs,

camalexin, and IAA (Figure 2A) (reviewed in Grubb and Abel,

2006). We noted that transcripts of HIG1/MYB51, encoding a

positive regulator of the indole-glucosinolate biosynthesis genes

(Gigolashvili et al., 2007), ATR4/CYP83B1/SUR2, encoding a

cytochrome P450 monooxygenase that channels IAOx toward

IGs (Barlier et al., 2000), and ST5a, encoding a sulfotransferase

implicated in Trp-derived glucosinolate biosynthesis (Piotrowski

et al., 2004), were more strongly induced by MeJA in jin1-9

than in wild-type plants (Figures 2A and 3A; see Supplemental

Table 1 online). By contrast, the glucosinolate catabolism gene

EPITHIOSPECIFIER PROTEIN (ESP), encoding the ESP, and

At2g39330, encoding a myrosinase putatively involved in gluco-

sinolate breakdown, were significantly less induced by MeJA in

jin1-9 than in the wild type (Figures 2A and 3A; see Supplemental

Table 1 online). To determine whether differential expression of

these genes leads to altered IG levels, we measured the levels

of three IGs in MeJA-treated and untreated plants of jin1-9,

wild type, and jin1-9 transformed with the E35S:MYC2 con-

struct (jin1-9/E35S:MYC2). These assays showed that the

concentrations of two IGs, indolyl-3-methyl glucosinolate and

4-methoxy-indolyl-3-methyl glucosinolate, were MeJA-responsive

(P < 0.01, two-way ANOVA); the latter was significantly higher in

MeJA-treated jin1-9 than in wild-type and jin1-9/E35S:MYC2

plants (Figure 3D), while the levels of 1-methoxy-indolyl-3-methyl

glucosinolate remained unchanged (data not shown).

We also found that the MeJA-responsive expression of MYB34/

ATR1 encoding a positive regulator of Trp and Trp-derived sec-

ondary metabolism genes was positively regulated by MYC2

(Figures 2A and 3A; see Supplemental Table 1 online). Although

this seems to be contradictory in light of the actual increases

observed at the IGs in the myc2/jin1 mutant, transcription of

MYB34/ATR1 is negatively regulated by IGs in a negative feed-

back loop (Smolen and Bender, 2002; Celenza et al., 2005); thus,

the reduced induction of MYB34/ATR1 by MeJA in the myc2/

jin1-9 background could indeed be consistent with higher IG

levels found in this mutant. Overall, these results suggest that

MYC2 is a negative regulator of the JA-dependent biosynthesis

of Trp-derived IGs in Arabidopsis.

In addition to IGs, the antimicrobial metabolite camalexin is

derived from IAOx (Figure 2A). The gene PAD3/CYP71B15,

which encodes a cytochrome P450 enzyme catalyzing the final

step in camalexin synthesis (Schuhegger et al., 2006), was

differentially expressed in the jin1-9 mutant following MeJA

treatment (Figure 2A; see Supplemental Table 1 online). Although

we did not determine camalexin levels in the MeJA-treated jin1-9

mutant, evidence from other studies shows that the increased

expression of PAD3 is associated with increased levels of

camalexin (Zhou et al., 1999), while the pad3 mutant displays

camalexin deficiency (Thomma et al., 1999). Camalexin is known

to be required in defense against necrotrophic fungal pathogens

(Thomma et al., 1999), and the possible increase of the levels of

this phytoalexin in the myc2/jin1 mutant might contribute to the

increased fungal disease resistance observed previously in this

mutant (Anderson et al., 2004; Lorenzo et al., 2004).

MeJA-Induced Auxin Biosynthesis in Arabidopsis

We hypothesized that the increased flux in the Trp-metabolic

pathway may also lead to alterations in IAA levels in MeJA-

treated plants. Differential expression of ATR4/CYP83B1/SUR1

in the myc2/jin1 mutant following MeJA treatment and the ap-

parent increase in IG levels observed here suggest that the in-

creased flux might be directed toward IGs and that this might

occur at the expense of IAA (Grubb and Abel, 2006). However,

because several genes involved in Trp biosynthesis were ex-

pressed at higher levels in response to MeJA in the myc2/jin1

mutant than in the wild type (Figure 2A), it is possible that IAOx

levels were also increased in the myc2/jin1 mutant. This could


lead to overall increases in IAA levels in MeJA-treated myc2/jin1

plants relative to those in wild-type plants. In addition, Trp-

and IAOx-independent but IGPS (for indole-3-glycerol phos-

phate synthase)-dependent IAA synthesis has been described

(Ouyang et al., 2000), and IGPS was also differentially expressed

in jin1-9 following MeJA treatment (Figures 2A and 3A; see Sup-

plemental Table 1 online). Therefore, we determined free IAA

levels in MeJA-treated and -untreated plants of jin1-9, wild type,

and jin1-9/E35S:MYC2. We found significantly increased levels

of IAA (P < 0.01, two-way ANOVA) (Figure 3E) in MeJA-treated

plants of all three lines relative to those in untreated plants, and

this is consistent with MeJA’s stimulatory effects on flux in the

Trp metabolic pathway. However, these assays did not reveal

any discernible difference in free IAA levels between the different

genotypes assayed (Figure 3E).

It should be noted, however, that in Arabidopsis, IAA can

also be synthesized through alternative pathways (reviewed in

Woodward and Bartel, 2005). Our gene expression assays showed

that the MeJA-responsive expression of ILR1, encoding an IAA-

amino hydrolyase that releases free IAA from the conjugated

forms (Bartel and Fink, 1995), was reduced in jin1-9 relative to

wild-type plants (Figure 2A; see Supplemental Table 1 online). Al-

though the relative contribution of Trp-derived and ILR1-mediated

IAA biosynthesis to final IAA levels in plant tissue is not known, it

is possible that the potential increase in IAA levels in myc2/jin1

through the activation of the Trp pathway might be negated by

the reduced expression of ILR1. Nevertheless, the increased IAA

level in MeJA-treated plants is a new finding and could contribute

to JA-mediated growth regulation.

MYC2 Is a Positive Regulator of JA-Mediated

Flavonoid Biosynthesis

MYC2, also known as RAP-1 (for R-homologous Arabidopsis

Protein-1), shows significant sequence similarity to R proteins,

regulating anthocyanin biosynthesis in maize (Zea mays) (de Pater

et al., 1997). Indeed, the development of anthocyanin in myc2/jin1

seedlings germinated in the presence of JA is abolished, while

strong anthocyanin pigmentation develops in JA-germinated

seedlings of MYC2-overexpressing plants (Lorenzo et al., 2004;

this study; data not shown). Furthermore, coronatine (a JA analog

and a phytotoxin produced by the bacterial pathogen P. syringae)–

induced anthocyanin content was found to be reduced signifi-

cantly in myc2/jin1 plants (Laurie-Berry et al., 2006). Together,

these results demonstrate that MYC2 is a positive regulator of the

JA-mediated anthocyanin biosynthesis in Arabidopsis, although

the molecular mechanism behind this observation is unknown.

Our large-scale gene expression analyses revealed that the

MeJA responsiveness of several genes involved in flavonoid

biosynthesis was reduced in the jin1-9 mutant relative to that in

wild-type plants (see Supplemental Figure 1 and Supplemental

Table 1 online). Among these are MYB75/PAP1 and EGL3, both

encoding positive regulators of flavonoid biosynthesis (Borevitz

et al., 2000; Zhang et al., 2003; Teng et al., 2005; Tohge et al.,

2005). In addition, the genes positively regulated by MYB75/

PAP1, such as PAL1, TT19/GST12, and UGT79B (Tohge et al.,

2005), with well-studied roles in flavonoid biosynthesis (Kitamura

et al., 2004; Rohde et al., 2004), showed differential expression

in the jin1-9 mutant (Figures 2A and 5A; see Supplemental Figure

1 and Supplemental Table 1 online). These results suggest that

MYC2, possibly acting by modulating the expression of the

positive regulators, MYB75/PAP1 and EGL3, positively regulates

flavonoid biosynthesis in Arabidopsis during JA signaling. Inter-

estingly, we found that the CAD gene involved in lignin biosyn-

thesis showed increased expression in the mutant. This is

consistent with the recent finding that a negative correlation

exists between flavonoids and lignin biosynthesis through the

phenylpropanoid pathway in Arabidopsis (Besseau et al., 2007).

MYC2 Is Required for the Sensitivity of Root Elongation to

Auxin Transport Inhibitors

Accumulating evidence suggests that flavonoids (e.g., anthocy-

anins) act as negative regulators of auxin transport (Brown et al.,

2001; Buer and Muday, 2004; Peer et al., 2004; Wasson et al.,

2006; Besseau et al., 2007). Auxin transport is required for primary

root elongation (Muday and Haworth, 1994; Jensen et al., 1998)

and plant growth (Besseau et al., 2007). Alterations in flavonoid

levels also modulate expression from genes encoding auxin

transporters (Lazar and Goodman, 2006). Our transcript profiling

experiments indicated that the JA-responsive expression of a

putative auxin efflux carrier family protein (At1g76520) was in-

creased in jin1-9 relative to wild-type plants (see Supplemental

Table 1 online). It is possible that the deficiency in MeJA-mediated

flavonoid synthesis in myc2/jin1 could alter auxin transport and

consequently lead to the reduced inhibition of primary root elon-

gation in plants grown in the presence of exogenous JA.

Naringenin, an early precursor in the flavonoid biosynthetic

pathway, inhibits primary root elongation as a result of its

inhibitory effects on auxin transport (Brown et al., 2001). Nar-

ingenin also complements the increased auxin transport pheno-

type of tt4 mutant plants with reduced flavonoids and auxin

transport (Brown et al., 2001). To determine whether MYC2 is

required for root naringenin sensitivity, we germinated seeds of

jin1-9, the wild type, and jin1-9/E35S:MYC2 in the presence of

naringenin and measured the primary root lengths (Figure 4A). The

roots of jin1-9/E35S:MYC2 were shorter than those of jin1-9 and

the wild type in the absence of naringenin. Although primary root

elongation was inhibited in all lines by naringenin and the combi-

nation of naringenin and MeJA, jin1-9/E35S:MYC2 roots were

hypersensitive (as shown by the percentages in Figure 4A) to

naringenin, MeJA, and the combination of both. We also tested

the sensitivity of jin1-9, wild-type, and jin1-9/E35S:MYC2 roots to

2,3,5-triiodobenzoic acid (TIBA), a synthetic auxin transport in-

hibitor. Again, the jin1-9/E35S:MYC2 roots were hypersensitive

(as shown by the percentages in Figure 4B) to TIBA in both the

presence and absence of exogenous MeJA. These results sug-

gest that MYC2 makes primary root elongation more sensitive to

inhibition by natural and synthetic auxin transport inhibitors and

therefore might have a role in regulating auxin transport. We

speculate that this effect of MYC2 might be due to MYC2’s

positive regulatory effects on JA-mediated flavonoid synthesis.

To further test the hypothesis that alterations in flavonoid levels

influence root elongation in the presence of JA, we examined the

sensitivity of max1 roots to MeJA. This mutant was selected

because, similar to MYC2, the MAX1/CYP71B1 gene product acts

2230 The Plant Cell

as a positive regulator of flavonoid synthesis and as a negative

regulator of auxin transport in Arabidopsis (Lazar and Goodman,

2006). Again, similar to MYC2, MAX1/CYP71B1 encoding a

cytochrome P450 affects flavonoid biosynthesis by modulating

the expression of the positive regulator MYB75/PAP1. As shown

in Figure 4C, we observed significantly reduced sensitivity of max1

roots to exogenously supplied MeJA, further suggesting a link

between MeJA-mediated flavonoid synthesis and auxin transport

that affects primary root elongation in Arabidopsis.

MYC2 Is Required for Oxidative Stress Tolerance

A link between JA and ascorbate biosynthesis and redox cycling

has been established (Sasaki-Sekimoto et al., 2005; Wolucka

et al., 2005). JA induces the expression of certain ascorbate

biosynthesis genes and the genes encoding (mono) dehydro-

ascorbate reductase (MDHAR and DHAR) involved in redox cy-

cling. Furthermore, treatment with JA or MeJA increases the de

novo synthesis of ascorbate together with DHAR and ascorbate

peroxidase activity (Sasaki-Sekimoto et al., 2005; Wolucka et al.,

2005). Together with anthocyanins, ascorbate is known to be

the main reactive oxygen species (ROS) scavenger in plants

(Nagata et al., 2003). We noted that the MeJA inducibility of

genes involved in oxidative stress tolerance was reduced in

jin1-9 relative to the wild type (Figure 5A; see Supplemental Table

1 online). In addition, TAT3, encoding a Tyr aminotransferase that

catalyzes the first step in the tocopherol (vitamin E) biosynthetic

pathway (Sandorf and Hollander-Czytko, 2002), showed re-

duced MeJA responsiveness in jin1-9 (Figure 5A; see Supple-

mental Table 1 online). Tocopherol is a JA- and stress-induced

chloroplast-located antioxidant that neutralizes photosynthesis-

derived ROS (reviewed in Munne-Bosch, 2005).

To determine whether MYC2 has a role in regulating oxidative

stress defenses during JA signaling, we treated wild-type, jin1-9,

and jin1-9/E35S:MYC2 lines with the superoxide generator

methyl viologen (Paraquat) after a pretreatment by MeJA for

6 h. Five days after methyl viologen treatment, 90% of jin1-9

plants were dead (Figure 5B). By contrast, only 5 and 15% of

treated jin1-9/E35S:MYC2 and wild-type plants, respectively,

were dead at this stage. The leaves of the majority of the

wild-type and jin1-9/E35S:MYC2 plants remained green (Figure

5B), and these plants subsequently recovered and produced

seed. In the absence of prior MeJA treatment, no differential ROS

Figure 4. MYC2 Is Required for Increased Sensitivity of Root Elongation

to Natural and Synthetic Auxin Transport Inhibitors.

(A) Root lengths of MeJA-treated and naringenin-treated (Nar)

10-d-old wild-type, jin1-9, and jin1-9/E35S:MYC2 Arabidopsis seed-

lings. Values (representative of two independent experiments) are

means of >30 seedlings for each treatment/genotype combination;

error bars denote SE. Values annotated with different letters are

significantly different (P < 0.01; Tukey’s LSD). Percentages of root

lengths of the different lines are relative to the respective untreated

controls.

(B) Root lengths of MeJA- and TIBA-treated 10-d-old wild-type, jin1-9,

and jin1-9/E35S:MYC2 Arabidopsis seedlings. Values (representative of

two independent experiments) are means of >30 seedlings for each

treatment/genotype combination; error bars denote SE. Values anno-

tated with different letters are significantly different (P < 0.01; Tukey’s

LSD). Percentages of root lengths of the different lines are relative to the

respective untreated controls.

(C) Root lengths of MeJA-treated 10-d-old wild-type and max1 Arabi-

dopsis seedlings. Values (representative of two independent experi-

ments) are means of >30 seedlings; error bars denote SE. Values

annotated with different letters are significantly different (P < 0.01;

Tukey’s LSD).


tolerance could be observed between the mutant and wild-type

plants (data not shown). Overall, these results are consistent with

the observation that MYC2 is a positive regulator of a subset of

JA-responsive genes involved in oxidative stress protection.

MYC2 Positively Regulates Resistance to Insect Herbivory

The JA signaling pathway is known to regulate many inducible

defenses effective against insects (for references, see Reymond

et al., 2004). To date, no transcriptional regulator of the JA

signaling pathway has been shown to alter insect tolerance in

Arabidopsis. MYC2, a gene that is responsive to insect herbivory

(Reymond et al., 2004), is a positive regulator of wound-responsive

genes such as VSP1, JR1, TAT, and LOX (Boter et al., 2004;

Lorenzo et al., 2004) that are also responsive to insect feeding.

Here, we identified additional JA- and insect-responsive genes

positively regulated by MYC2 (Figure 6A; see Supplemental

Table 1 online). At least two of these genes with reduced MeJA

Figure 5. MYC2 Positively Regulates Oxidative Stress Tolerance in a JA-Dependent Manner.

(A) Q-RT-PCR expression analysis of anthocyanin- and ascorbate-related genes. See Figure 3A legend for details of Q-RT-PCR.

(B) Arabidopsis plant phenotypes at 4 d after treatment with 50 mM methyl viologen. Plants were pretreated for 6 h with 0.1 mM MeJA. Photographs are

representative of four independent experiments each with 20 plants per genotype/treatment combination.

2232 The Plant Cell

responsiveness in the jin1-9 mutant have demonstrated anti-

insect activities. VSP2 encodes an anti-insect acid phosphatase

enzyme (Liu et al., 2005), and the At4g08870 locus encodes an

ortholog of the tomato (Solanum lycopersicum) arginase that

reduces larval weight gain by degrading the essential amino acid

Arg in the herbivore midgut (Chen et al., 2005). The reduced

expression of these genes in the myc2/jin1 mutant suggests that

the insect resistance might be reduced in the mutant. Interest-

ingly, however, as we reported above, we found increased levels

of IGs in the myc2/jin1 mutant (Figure 3D). This might suggest

otherwise—that is, the myc2/jin1 mutant may be more tolerant of

insects, as IGs are often implicated in insect defense (Wittstock

and Halkier, 2002). Therefore, we investigated whether insect

herbivory is altered in the myc2/jin1 mutants. No-choice feeding

experiments were set up with the generalist herbivore Helicoverpa

armigera (cotton bollworm or tobacco budworm). We found that

the weight gain of neonate larvae feeding on MeJA-pretreated

jin1-9 plants was significantly higher than that on similarly treated

wild-type and jin1-9/E35S:MYC2 plants after 6 d of feeding (Fig-

ure 6B). These results show that MYC2 function is required for

JA-mediated tolerance to H. armigera in Arabidopsis.

MYC2 Preferentially Binds to an Extended G-Box Motif

The large number of genes that displayed MYC2 dependence for

their MeJA-responsive expression prompted us to quantitatively

determine the optimal DNA binding site of MYC2. Determination

of the optimal binding site can be of value to identify genes that

may be regulated by MYC2 at the transcription level, thus making

it possible to construct the potential MYC2 regulon. Previous

reports indicated that MYC2 can bind to the G-box–related

hexamers 59-CACNTG-39 (de Pater et al., 1997), 59-CACATG-39

(Abe et al., 1997), and 59-(T/C)ACGTG-39 (Yadav et al., 2005).

However, these binding sites were determined in a nonquanti-

tative and biased way using selected specific DNA sequences.

Here, we opted for an unbiased and quantitative method (Xue,

2005) to identify the preferred DNA binding sites of MYC2.

Briefly, a purified MYC2-CelD-6xHis fusion protein was used

for sequential steps of affinity selection of binding sequences

from a pool of biotinylated random sequence oligonucleotides

(30-mers) (Xue, 2005). The 6xHis-tagged cellulase (CelD) allows

for affinity purifications (on cellulose or Ni) and quantification of

the binding of selected oligomers to the MYC2 fusion protein

(CelD as an enzymatic reporter). After the third and fourth

selection rounds in the purification process, a massive increase

in DNA binding activity, indicative of a strong enrichment for

MYC2 binding sites in the oligonucleotide pools, was observed

(data not shown). The oligonucleotides from the third and fourth

selection round were cloned, and 40 clones from each pool were

sequenced. Overall, the majority of these clones contained at

least one CACGTG palindromic hexamer (G-box), suggestive of

the G-box being the preferred MYC2 core binding site (Figure

7A). Some of the sequenced oligonucleotides contained the

G-box–related motifs 59-CACATG-39 and 59-CACGTT-39. In to-

tal, 38 of the sequenced clones were amplified by PCR with

biotinylated primers, the products purified, and their MYC2

binding activity measured. In Figure 7A, these oligonucleotides

are ranked according to their MYC2 binding capacity. By and

large, oligomers containing the G-boxes had the strongest MYC2

binding capacity, followed by those with the 59-CACATG-39 and

59-CACGTT-39 motifs.

The core G-box is a sequence element present at least once in

nearly 30% of the 59 upstream regions of all Arabidopsis genes

(data not shown). Given the abundance of this sequence as well

as the presence of many other bHLH proteins that can potentially

bind to this sequence, it is likely that not all G-box–containing

genes are regulated by MYC2. Therefore, we further defined

the optimal DNA binding sequence of MYC2. Alignment of the

palindromic G-boxes revealed additional conserved bases in the

sequences that flank the G-box hexamer (Figure 7B). To deter-

mine whether these conserved flanking sequences contribute to

the MYC2 DNA binding capacity, synthetic oligonucleotides

were obtained with mutations in these regions (Figure 7C). Re-

markably, all mutations introduced into these flanking nucleo-

tides reduced the MYC2 DNA binding capacity of the D27

oligonucleotide. Both the upstream (positions 1 to 3 in Figure 7B)

Figure 6. MYC2 Positively Regulates Resistance to H. armigera Herbiv-

ory during JA Signaling.

(A) Q-RT-PCR expression analysis of insect resistance and wound

response genes. See Figure 3A legend for details of Q-RT-PCR.

(B) Average weight of H. armigera larvae at 6 d after neonate larvae were

placed on 5-week-old Arabidopsis plants. Plants were pretreated for 24 h

with 0.5 mM MeJA. Data are means of 15 individual plants challenged

with five neonate larvae each; error bars denote SE. Values annotated with

different letters are significantly different (P < 0.01; Tukey’s LSD).


Figure 7. MYC2 Preferentially Binds to an Extended G-Box Motif.

(A) Sequences and MYC2 binding activities of 38 30-mers from affinity purification selection rounds 3 and 4. MYC2 binding activities for different

sequences are expressed relative to the highest binding activity (relative binding activity) observed in D27. Values are means of three replicates; error

2234 The Plant Cell

and downstream (positions 16 to 18 in Figure 7B) T-rich regions

had a significant effect on the binding activity of MYC2. Mutation

of the conserved pyrimidine at position 13 (Figure 7B) to a purine

only slightly reduced the binding activity. More importantly, mu-

tations introduced into the core G-box palindrome (59-CAC-

ATG-39 or 59-CACGTT-39) drastically diminished MYC2 binding

activity (Figure 7C). A position–weight matrix based on the

alignment shown in Figure 7B was then used in a stringent in

silico screening for the presence of strong MYC2 binding sites in

the 59 upstream regions of Arabidopsis genes (see Supplemental

Table 2 online). The screening revealed that the set of 778

MYC2-regulated genes is enriched for strong MYC2 binding

sites compared with the whole Arabidopsis genome (8.0 versus

4.2%, respectively; P < 0.01, hypergeometric test). This enrich-

ment was especially evident when MYC2-regulated TFs were

compared with the whole Arabidopsis TF complement (20 versus

8.9%; P < 0.01) (see Supplemental Table 2 online). After clus-

tering of the motifs in the upstream regions of the MYC2-

regulated genes, 10 representative CACGTG core motifs were

assessed for their MYC2 binding capacity (Figure 7D). These in-

cluded the motifs found in the promoters of two MYC2-regulated

TF genes (i.e., ERF4 and ERF1) as well as in the promoter of

MYC2. All of these Arabidopsis motifs displayed significant

MYC2 binding activity except the one present in the upstream

region of At4g22212.

Evidence That Negative Regulation of PDF1.2 by MYC2 Is

Mediated by Suppression of ERF1

Current models predict a direct mutual antagonism of MYC2 and

ERF1 on the expression of pathogen defense response genes

such as PDF1.2 and PR4/HEL and wound response genes such

as VSP and LOX (Lorenzo et al., 2004; Lorenzo and Solano,

2005). It was speculated that MYC2 might directly bind to the

PDF1.2 promoter to suppress its expression (Lorenzo et al.,

2004). The PDF1.2 promoter region contains a G-box–like motif,

59-CACATG-39 (Brown et al., 2003). This motif, depicted as

PDF1.2-RD22 in Figure 7D, is the same as the motif implicated as

a MYC2 binding motif in the RD22 promoter during abscisic

acid– and drought-responsive expression of RD22 (Abe et al.,

1997). This G-box–like motif differs from the core of the optimal

MYC2 binding site by at least one nucleotide (G). In our DNA

binding experiments shown in Figure 7D, this motif and flanking

sequences did not display any MYC2 binding capacity at all,

suggesting that MYC2 might not interact directly with the PDF1.2

promoter. Interestingly, the promoter region of ERF1, a gene

involved in the ET- and JA-dependent induction of PDF1.2

(Lorenzo et al., 2003), contains both a G-box (ERF1-GBOX) and

a 59-CACATG-39 motif, depicted as ERF1-RD22 in Figure 7D. We

found that only the ERF1-GBOX had significant MYC2 binding

affinity, most likely increased by the flanking T-rich regions and a

C (pyrimidine) immediately downstream of the core hexamer

(Figure 7D). These results, together with the increased MeJA

responsiveness of ERF1 (see Supplemental Figure 1 and Supple-

mental Table 1 online) in myc2/jin1, suggest that the negative

regulation of PDF1.2 expression by MYC2 is most likely mediated

through the negative regulation of transcriptional activators of

PDF1.2 such as ERF1.

MYC2 Negatively Regulates Its Own Transcription

As shown in Figure 7D, the upstream region of the MYC2 gene

contains a MYC2 binding site with a significant binding capacity,

suggestive of a potential autoregulatory loop for MYC2 tran-

scription. To investigate this possibility further, we comparatively

analyzed MYC2 transcript levels in the presence or absence of

MeJA. In these experiments, we used a specific PCR primer pair

(59 untranslated region [UTR]) to distinguish the wild type and the

mutant MYC2 alleles from the transgenic allele in the comple-

mented line (Figure 8B). Both the 59UTR and the MYC2 primer

pairs performed similarly for the wild type and the mutant MYC2

alleles under both conditions tested. Also, MYC2 expression, as

detected by the MYC2 and 59UTR primer pairs, was clearly

induced by MeJA in both the wild type and jin1-9, while MYC2

was constitutively expressed in jin1-9/E-35S:MYC2 (Figure 8A).

However, expression of the MYC2 mutant allele detected using

the 59UTR primer pair was reduced significantly in the comple-

mented line (jin1-9/E-35S:MYC2) compared with the mutant

background, whereas the E35S:MYC2 transgene, as detected

by the MYC2 primer pair, remained highly expressed in the

complemented background. This result suggests that MYC2 is

capable of negatively regulating its own expression. To rule out

any positional insertion effects of the transgene, several inde-

pendent homozygous complemented lines were analyzed, and

they all performed similarly (data not shown). In addition, we

found that this negative regulation was not sensitive to cyclo-

heximide (CHX) (Figure 8C), suggesting that new protein syn-

thesis is not required for MYC2’s negative regulatory effects on

its own transcription. Given that multiple biotic and abiotic stress

factors induce MYC2 expression, it is tempting to speculate that

Figure 7. (continued).

bars denote SD. Gray boxes, G-box; black boxes, 59-CACATG-39; white boxes, 59-CACGTT-39. Motifs at the edges of the 30-mers are completed by the

sequences from the flanking regions of the random sequence oligonucleotide pool used for binding site selection (TAGC at the 59 end and GCTG at the

39 end; see Xue (2005) for complete sequences of flanking regions SP-A and SP-S1).

(B) Alignment of G-boxes and flanking sequences of MYC2-selected motifs containing a single CACGTG box with relative DNA binding activity of >30%

of the highest affinity oligonucleotide (D27) (see [A]). Black boxes, 100% conserved; gray boxes, 75% conserved. The illustration depicting this

alignment was created with WebLogo (Crooks et al., 2004).

(C) Sequences and MYC2 binding activities of D27-derived synthetic oligonucleotides. Binding activities of MYC2 and shading are as in (A), except for

the white boxes denoting mutations from the original D27 sequence.

(D) Sequences and MYC2 binding activities of motifs present in Arabidopsis promoter regions. Probes are synthetic oligonucleotides. The binding

capacity of MYC2 is expressed as in (A).


this negative autoregulation capability might be a mechanism

that contributes to the fine-tuning of the signaling pathways by

controlling MYC2 levels.

MYC2 Modulates the JA-Dependent Transcription

of TF Genes

Many of the MYC2-regulated genes identified through transcript

profiling and the subsequent Q-RT-PCR encode TFs (see Sup-

plemental Figure 1 and Supplemental Table 1 online). A signif-

icant enrichment for strong MYC2 binding sites was found in the

upstream regions of MYC2-regulated TF genes (see Supple-

mental Table 2 online). DNA binding assays shown in this report

as well as in previous publications (de Pater et al., 1997; Abe et al.,

2003; Boter et al., 2004; Yadav et al., 2005) clearly demonstrated

that MYC2 can bind to the CACNGT core motif. Furthermore, our

additional promoter analyses of a subset of MYC2-regulated TFs

(given in Figure 10B) for the presence of CACGTG and CACATG

motifs using the Arabidopsis Gene Regulatory Information Centre

database (Palaniswamy et al., 2006) revealed that 82 and 88% of

such TFs, respectively, had at least one of these core motifs in

their promoters. Moreover, 71% of these TFs had at least one

copy of both of these motifs in their promoters. Therefore, strong

enrichment of these motifs in the promoters of MYC2-regulated

TFs might suggest a hierarchical model in which MYC2 positively

or negatively modulates the JA-dependent transcription of other

TF genes, which in turn might control the JA-dependent transcrip-

tion of the downstream JA response genes. In this model, MYC2

would be positioned relatively upstream in the JA signal trans-

duction pathway, possibly downstream from COI1 (Lorenzo et al.,

2004) and MKK3 and MPK6 mitogen-activated protein kinase

pathways (Takahashi et al., 2007) but upstream from MYC2-

regulated TFs. Significant functional overlaps observed for rela-

tively large numbers of genes found to be differentially expressed

in myc2/jin1 (this study), coi1 (Devoto et al., 2005), and 35S:ERF1

plants (Lorenzo et al., 2003) are consistent with this proposal.

To explore the possibility that MYC2 modulates JA-responsive

gene expression through MYC2-dependent TFs, we obtained

several homozygous T-DNA insertion lines for the following

MYC2-regulated TF genes; ERF2, ERF6, ERF11, WRKY26,

WRKY33, MYB51, MYB109, At1g33760, and ZAT10. An exten-

sive Q-RT-PCR expression study was then set up to determine

whether the expression of MYC2-regulated genes is affected in

these mutant backgrounds in response to MeJA treatment.

As shown in Figure 9A, the expression profiles of pathogen

defense–related genes (cluster I) and wound response/insect

Figure 8. MYC2 Directly and Negatively Regulates Its Own Expression.

(A) Expression from the wild type, mutant, and transgenic MYC2 alleles was comparatively examined using 59UTR and MYC2 Q-RT-PCR primer pairs in

mock- and 0.1 mM MeJA–treated plants of Col-0, jin1-9, and jin1-9/E-35S:MYC2. Note that as shown in (B), the MYC2 primer pair binds to the mutant

MYC2 allele upstream from the T-DNA insertion site and detects similar transcript levels as found in the wild type. Error bars denote SE.

(B) Schematic illustration of the binding regions of the 59UTR and MYC2 primers on the wild type, mutant, and both mutant and transgenic MYC2 alleles

on wild-type, jin1-9, and jin1-9/E35S:MYC2 plants, respectively. Note that there is no 59UTR binding site on the E35S:MYC2 construct.

(C) Expression detected from the jin1-9 and E35S:MYC2 alleles by the MYC2 primer pair in CHX-treated and CHX- and MeJA-treated jin1-9 and jin1-9/

E-35S:MYC2 plants. See text for details. Data are means of three biological replicates (more than five pooled plants each). Error bars denote SE. Values

annotated with different letters in (A) and (C) are significantly different (P < 0.01; Tukey’s LSD).

2236 The Plant Cell

Figure 9. MYC2-Regulated TFs Modulate the Expression of MYC2-Regulated Genes.


resistance genes (clusters III and IV) suggest that these groups of

genes are coregulated generally in an antagonistic manner. For

instance, like ERF1 and in contrast with MYC2, WRKY33 acts as

a negative regulator of wound response/insect resistance genes

and as a positive regulator of pathogen defense–related genes

during JA signaling. Similar to MYC2, ERF11 and At1g33760

downregulate pathogen defense–related genes and ERF2 acti-

vates wound response/insect resistance genes in JA-treated

plants. MYB51 activates IGs biosynthesis genes in a JA-dependent

manner. ZAT10, ERF4, and WRKY26 act as negative regulators

of basal transcript levels of the majority of the genes in the

absence of MeJA treatment. At least two of these TFs (ZAT10

and ERF4) contain an EAR-repression domain (Kazan, 2006).

This repression seems to disappear after MeJA treatment, al-

though the negative regulatory effect of WRKY26 on the cluster

IV genes is enhanced.

We also examined the expression profiles of different MYC2-

regulated TFs in this panel of mutants (Figure 9B). Examples of

cross-regulation between MYC2-regulated TFs are the down-

regulation of ERF4, TDR1, MYB34/ATR1, and At1g06160 and the

upregulation of ERF1 and WRKY26 by WRKY33, the down-

regulation of EGL3, MYB34/ATR1, and MYB109 by WRKY26, and

the upregulation of At1g06160 and MYB109 by ERF2. Interest-

ingly, basal transcript levels of MYC2 seem to be repressed by

ERF6, ERF11, and ZAT10. These expression profiles are illustra-

tive of the regulatory complexities downstream of MYC2.

In an effort to better define the relative position of MYC2 within

the JA signaling pathway, we wanted to know whether MYC2 is a

primary JA response gene. The data shown in Figure 8 indicated

that in the presence of CHX, the MeJA inducibility of MYC2 was

abolished. Because de novo protein synthesis is not required for

the induction of primary response genes by JA (van der Fits and

Memelink, 2001; Pauw and Memelink, 2004), this observation

indicates that, by definition, MYC2 is not a primary JA response

gene. In additional experiments, we examined MYC2, ERF1,

PDF1.2, and VSP1 expression in wild-type plants treated with

MeJA, CHX, or both. CHX treatment significantly induced MYC2,

ERF1, and VSP1 expression, and this induction was severalfold

higher than that by MeJA (Figure 10A). By contrast, CHX treatment

significantly suppressed PDF1.2 expression. In the presence of

the protein translation inhibitor CHX, the MeJA inducibility of

ERF1, PDF1.2, and VSP1 was abolished, as observed for MYC2

(Figure 10A). These experimental results are consistent with the

model (Pauw and Memelink, 2004) proposing that, by definition,

MYC2, ERF1, PDF1.2, and VSP are all secondary JA response

genes requiring the synthesis of upstream regulators.

Next, we asked whether TF genes showing differential ex-

pression in myc2/jin1 are direct or indirect targets of MYC2. We

compared the expression of these TFs in CHX- and MeJA-

treated jin1-9 and jin1-9/E35S:MYC2 based on the view that the

existing levels of MYC2 should be sufficient to modulate the

expression from primary target genes. By contrast, new protein

synthesis would be required for the JA-dependent expression of

secondary target genes (van der Fits and Memelink, 2001; Wang

et al., 2005). Similar to MYC2 and ERF1, the CHX treatment alone

substantially induced all TF genes (data not shown), suggesting

that the expression of these genes might be blocked by contin-

uously synthesized repressors. Among the TF genes examined,

MYB34/ATR1, MYB75, and ZAT10 showed differences between

CHX- and MeJA-treated plants of jin1-9 and jin1-9/E35S:MYC2

(Figure 10B). The MeJA-inducible differential expression of the

remaining TFs in the mutant could not be observed in the

presence of CHX, possibly due to the superinducibility of these

genes by CHX treatment alone.

DISCUSSION

The results described here give MYC2 a central role within the JA

signaling pathway in regulating diverse JA responses. Together

with prior observations of MYC2 mediating crosstalk between

JA–abscisic acid and JA–salicylic acid signaling, a novel role

implicating MYC2 in auxin transport also indicates that MYC2

is a key junction point in a broader network involving multiple

hormone signaling pathways.

Recently, JA signaling has been implicated in mediating the

long-distance information transmission leading to a systemic

immunity in Arabidopsis (Truman et al., 2007). Indeed, in the

systemic tissue of plants challenged with avirulent bacterial or

fungal pathogens, JA biosynthesis genes along with the genes

involved in the synthesis of aromatic amino acids and glucosi-

nolate and phenylpropanoid metabolism genes are induced

(Schenk et al., 2003; Truman et al., 2007). Interestingly, the JA-

mediated systemic defense against a virulent strain of P. syringae

was compromised in the unchallenged leaves of the myc2/jin1

mutant, which was locally treated with an avirulent strain (Truman

et al., 2007), suggesting that MYC2 function is required for JA-

mediated systemic resistance against bacterial pathogens.

The results presented here clearly show that MYC2 can indeed

positively or negatively regulate many JA-dependent functions

mentioned above. The differential effects of MYC2 on different

JA responses might be due to the fact that precise coordination

of these responses might be required for resource management

and during adaptation to challenge by biotic and abiotic stress

factors. For instance, although both pathogen and insect attacks

stimulate JA biosynthesis, most changes in JA-responsive gene

expression occur in an attacker-dependent manner (De Vos et al.,

2005), suggesting that plants can divert limited resources in the

best possible way. Therefore, one of the important functions of

Figure 9. (continued).

(A) Expression profiles of MYC2-regulated response/end point genes in mutant lines of MYC2-regulated TFs show clusters of coregulated genes.

Samples were treated for 6 h with 0.1 mM MeJA (or mock controls). Data are means of three biological replicates (>20 pooled plants each) and are

expressed as ratios of expression levels in the mutant lines to expression levels in the wild type. Clustering was done by complete linkage of Euclidian

distances. Clusters of coregulated genes (I to IV) are shown in red at right, and the red line at left marks the cutoff distance used for the clustering.

(B) Expression profiles of MYC2-regulated TF genes in mutant lines of MYC2-regulated TFs illustrate cross-regulation between different TFs. Samples

and data are as in (A).

2238 The Plant Cell

MYC2 might be to coordinate JA-dependent defense responses

by positively and negatively regulating JA-responsive insect and

pathogen defense genes, respectively. Indeed, the myc2/jin1

mutant shows increased resistance to fungal and bacterial

pathogens but, as we have shown here, reduced resistance to

an insect pest.

One would expect that MYC2 expression itself should be

tightly controlled at the transcriptional level during JA signaling

for precise and rapid coordination of diverse JA-dependent

responses. Indeed, Takahashi et al. (2007) proposed that there

might be two separate JA-dependent pathways regulating MYC2

expression. One of these pathways is dependent on MKK3 and

MPK6 and negatively regulates MYC2, while the other is inde-

pendent from these mitogen-activated protein kinase pathways

and positively regulates MYC2. Our results presented here also

indicate that MYC2 is capable of negatively regulating its own

expression, possibly by binding directly to the G-box found its

promoter. The fact that this negative autoregulation is consis-

tently observed in multiple jin1-9/E35S:MYC2 lines, while the

opposite is not observed in jin1-9, suggests that this might be a

mechanism operating when MYC2 levels reach a critical thresh-

old, such as in plants simultaneously exposed to multiple biotic

and/or abiotic stress conditions. Nevertheless, our findings,

together with those by Takahashi et al. (2007), imply that both

negative and positive regulation play roles in the control of MYC2

expression and that this might be an important fine-tuning

mechanism of the JA signaling pathway.

Our results from microarray experiments and subsequent func-

tional analyses showed that in the absence of MeJA treatment,

very few changes were observed in gene expression and phe-

notypic responses between the wild type, myc2/jin1, and jin1-9/

E35S:MYC2. This suggests that additional factors induced and/

or activated by MeJA might also be required for MYC2 action

(Lorenzo et al., 2004). A plausible explanation, therefore, would

be that JA activates MYC2 at the posttranscriptional level. In-

deed, a reversible protein phosphorylation step is required for

JA-mediated gene expression, as JA-dependent gene induction

through this pathway was abolished in both coi1 and myc2/jin1

(Rojo et al., 1998). However, to date, the phosphorylation of MYC2

has not been demonstrated.

The large number of genes that showed some degree of

significant MYC2 dependence for their JA-responsive expres-

sion might exclude the possibility that MYC2 directly controls

the transcription of all of these genes. It is possible that MYC2

directly and indirectly regulates the JA-dependent transcription

of a set of TFs, which in turn regulate the transcription of the

secondary JA response genes. Several lines of evidence support

this view. First, MYC2-regulated TFs are enriched for the pres-

ence of strong MYC2 binding sites in their promoter regions (see

Supplemental Table 2 online). Furthermore, several JA-dependent

TF genes show differential expression in the jin1-9 mutant (see

Supplemental Figure 1 online), and recent research suggests

that some of these TFs can indeed regulate subsets of genes

and phenotypes also regulated by MYC2 itself. For instance,

comparison of our microarray data on jin1-9 with that on ERF1-

overexpressing plants (Lorenzo et al., 2003) showed that a

number of genes negatively regulated by MYC2 were positively

regulated by ERF1. This includes not only defense genes but also

other functional categories such as Trp biosynthesis genes.

Recent work has also shown that overexpression of the MYC2-

repressed TF genes ERF1, ERF2, TDR1, WRKY33, and ERF6

resulted in increased resistance to fungal pathogens such as

B. cinerea and F. oxysporum (Berrocal-Lobo and Molina, 2004;

Gutterson and Reuber, 2004; McGrath et al., 2005; Zheng

et al., 2006b; C. Edgar and K. Kazan, unpublished data). Indeed,

myc2/jin1 shows increased resistance to all of these pathogens

(Anderson et al., 2004; Lorenzo et al., 2004; Nickstadt et al.,

2004; Laurie-Berry et al., 2006). Similarly, it was previously shown

that overexpression of the MYC2-regulated TFs MYB75/PAP1

and EGL3 with reduced expression in the myc2/jin1 mutant

results in increased flavonoid biosynthesis (Tohge et al., 2005),

suggesting that MYC2’s effects on JA-mediated flavonoid metab-

olism are partially mediated by these TFs. The myc2/jin1 mutant

also shows increased resistance to F. oxysporum (Anderson

et al., 2004), and as we found here, ERF2 was upregulated in the

mutant. ERF2 encodes a positive regulator of JA-responsive

defense genes, and overexpression of this TF leads to increased

Figure 10. MYC2 Is a Secondary JA Response Gene.

(A) MeJA-, CHX-, and MeJA- and CHX-mediated expression of MYC2,

PDF1.2, ERF1, and VSP1. Note that relative expression level on the y axis

is given logarithmically. Data are means of three biological replicates.

Error bars denote SE. Values annotated with different letters are signif-

icantly different (P < 0.01; Tukey’s LSD).

(B) MYC2-modulated TF gene expression in CHX- and MeJA-treated

plants of jin1-9 and jin1-9/E35S:MYC2. Please note that relative expression

level on the y axis is given logarithmically. See Figure 3A legend for details

of Q-RT-PCR and Methods for details of treatments. Error bars denote SE.


F. oxysporum resistance in transgenic plants (McGrath et al.,

2005). Moreover, our gene expression analyses in T-DNA lines of

MYC2-modulated TFs suggest that these TFs might indeed

regulate an overlapping subset of MYC2-modulated genes (Fig-

ures 9A and 9B). Further functional analyses of TFs directly or

indirectly regulated by MYC2 should provide additional insights

into the fine regulation of the different JA responses at the

transcriptional level.

Some other effects of MYC2 on transcription could also occur

as a result of MYC2-modulated changes in metabolite levels,

such as altered flavonoid accumulation, changes in phytohor-

mone balance (Nickstadt et al., 2004; Laurie-Berry et al., 2006), or

altered redox status (Figure 5). For instance, our gene expression

analyses (see Supplemental Figure 1 and Supplemental Table

1 online) suggest that MYC2 might modulate ET and JA levels by

negatively and positively regulating the ET and JA biosynthesis

genes ACS6 and AOC4, respectively, during JA signaling.

Importantly, our results presented here show that MYC2

negatively regulates the Trp metabolic pathway during JA sig-

naling. One class of Trp-derived secondary metabolites is the

IGs. Our results are consistent with several reports that show that

Arabidopsis IGs are elevated by treatment with MeJA, Erwinia

carotovora elicitors, or Phytium sylvaticum and that an intact JA

signaling cascade is required for their induction (Brader et al.,

2001; Mikkelsen et al., 2003; Bednarek et al., 2005; Sasaki-

Sekimoto et al., 2005). However, the transcriptional control of

JA-mediated IG biosynthesis is not well known. Here, we showed

that MYC2 is a negative regulator of JA-mediated IG biosynthe-

sis, and again, this effect is likely to be at least partially mediated

by the negative regulation of positive regulators of this pathway.

Indeed, we found that the expression of HIG1/MYB51, encoding

a positive regulator of this pathway, was increased in the myc2/

jin1 mutant. A recent report showed that HIG1/MYB51 activates

IG biosynthesis genes such as TSB1, ATR4/CYP83B1/SUR2,

and ST5a (Gigolashvili et al., 2007). Remarkably, both HIG1/

MYB51 and its downstream targets showed increased expres-

sion in the myc2/jin1 mutant (Figure 2A). In addition, our expres-

sion analysis showed reduced expression of IG biosynthesis

genes such as ASB, TSA1, TSB2, IGPS, ST5a, and ATR4/

CYP83B1/SUR2 in the JA-treated myb51 mutant (Figure 9A).

Intact IGs and glucosinolates, in general, are thought to be

nontoxic, but their breakdown products, isothiocyanates and

nitriles, can be toxic (reviewed in Wittstock and Halkier, 2002;

Grubb and Abel, 2006). Breakdown of glucosinolates to their

nitrile derivatives is mediated through the ESP (Lambrix et al.,

2001; Zabala et al., 2005). In the absence of ESP (Lambrix et al.,

2001) or under conditions in which ESP expression is reduced

(e.g., in a myc2/jin1 mutant), glucosinolates spontaneously de-

grade to their respective isothiocyanate derivatives. Arabidopsis

isothiocyanates have demonstrated in vitro (Olivier et al., 1999;

Brader et al., 2001, 2006; Tierens et al., 2001) and in planta

(Tierens et al., 2001; Brader et al., 2006) antimicrobial properties.

Therefore, part of the enhanced disease resistance in myc2/jin1

could be mediated by directing IG breakdown toward isothio-

cyanates. IGs are also involved in defense against certain insect

pests. Despite increased levels of IGs, the myc2/jin1 mutant

showed reduced tolerance to H. armigera. However, we also

observed reduced expression of wound and insect defense

genes such as VSP1, VSP2, At4g08870 (arginase), and ADC2/

SPE2 in the jin1/myc2 mutant during JA signaling. This suggests

that these insect defensive proteins might have a role in defense

against H. armigera.

The potential of JA to induce auxin biosynthesis was originally

proposed by Devoto et al. (2005). Here, we show experimentally

that MeJA treatment can indeed increase IAA levels and that this

could contribute to the MeJA-mediated growth regulation. This

increase is probably due to the activation of both Trp-dependent

and Trp-independent IAA biosynthesis (e.g., via ILR1). Plants

overexpressing ERF1 show both increased expression of genes

encoding Trp biosynthetic enzymes and increased inhibition

of root elongation by JA (Lorenzo et al., 2003), indicating that

auxin homeostasis might also be altered in ERF1-overexpressing

plants grown in the presence of exogenous JA. Interestingly, it

was also shown that auxin increases the transcript levels of

JA biosynthesis genes in Arabidopsis (Tiryaki and Staswick,

2002), suggesting that a positive feedback loop regulates these

hormone levels. MeJA-mediated IAA synthesis may be critical

for the proper regulation of plant growth and development

under biotic stress. Indeed, a recent study in insect-attacked

tobacco (Nicotiana tabacum) plants suggests that JA signaling

suppresses regrowth and contributes to apical dominance, a role

expected from auxin (Zavala and Baldwin, 2006). A similar role

for auxin was also proposed for ET-mediated inhibition of

root elongation (Rahman et al., 2001; Stepanova et al., 2005).

ET inhibits root elongation through upregulation of the Trp

biosynthesis genes ASA1 and ASB1, which presumably leads

to the accumulation of inhibitory levels of auxin in the root tip

(Stepanova et al., 2005).

We also provided evidence that MYC2 is a positive regulator of

enzymes and regulators involved in JA-mediated flavonoid bio-

synthesis. Flavonoids are recognized as endogenous regulators

of auxin transport (Besseau et al., 2007, and references cited

therein). Our experiments showed that jin1-9/E35S:MYC2 roots

were hypersensitive to the auxin transport inhibitors naringenin

and TIBA. In addition to the previously known JA-insensitivity

phenotype, the myc2/jin1 roots exhibit increased resistance to

the phytotoxin coronatine (Laurie-Berry et al., 2006). Both JA and

coronatine induce flavonoid biosynthesis in wild-type plants, but

this was compromised in the myc2/jin1 mutant (Lorenzo et al.,

2004; Laurie-Berry et al., 2006). Thus, we propose that the

increased and reduced sensitivities of the jin1-9/E35S:MYC2

and myc2/jin1 roots, respectively, to exogenous JA might be due

to altered flavonoid levels affecting auxin transport. A recent

study by Zheng et al. (2006a) showed that, similar to JA, bestatin,

an amino peptidase inhibitor, specifically activates the JA sig-

naling pathway, induces MYC2, and inhibits root elongation in

Arabidopsis. Remarkably, the myc2/jin1 mutant shows reduced

sensitivity to the bestatin-mediated inhibition of root elongation

(Zheng et al., 2006a). Although the possible reason(s) for bestatin-

mediated inhibition of the root elongation phenotype was not

examined by Zheng et al. (2006a), previous studies showed that

bestatin blocks auxin transport in a manner similar to flavonoids

(Murphy et al., 2000).

The JA signaling pathway is known to modulate ozone-induced

cell death in Arabidopsis, possibly by regulating ROS homeo-

stasis. Most, if not all, JA signaling and biosynthetic mutants,

2240 The Plant Cell

including jar1, coi1, fad3/fad7/fad8, oji1, and opr3, show in-

creased ozone sensitivity (Kanna et al., 2003; Overmyer et al.,

2003; Sasaki-Sekimoto et al., 2005). In addition, exogenous ap-

plication of JA alleviates lesion formation in the ozone-sensitive

rcd1 mutant (Kanna et al., 2003; Overmyer et al., 2003). Here, we

demonstrate that MYC2 is also a regulator of different MeJA-

mediated antioxidant defenses. The decrease in oxidative stress

tolerance of the myc2/jin1 mutants under MeJA treatment is

likely due to the combined effect of the reduced expression of

genes associated with anthocyanin and tocopherol biosynthesis

and ascorbate recycling.

Despite its quantitative effects on diverse JA-dependent pro-

cesses, our results also indicate that MYC2 is not a primary JA

response gene (see Pauw and Memelink, 2004, for further

discussion). Indeed, recent studies showed that MYC2 acts

downstream from COI1 and mitogen-activated protein kinase

pathways in the JA signaling pathway (Lorenzo et al., 2004;

Takahashi et al., 2007). Nevertheless, MYC2 probably acts re-

latively upstream within the secondary JA signaling cascade to

affect the diverse JA-dependent phenotypes described here.

Our results presented here also indicate that in the presence of

CHX, MYC2 dependence could be observed in only a few TFs.

The data presented in Figure 7D show that MYC2 binds strongly

to the conserved sequence motifs found in the promoters of

both ERF1 and ERF4, suggesting that these genes are direct

MYC2 targets. However, in the presence of CHX, differential

expression of these genes by MeJA in jin1-9 could not be

observed. However, we found that almost all MYC2-modulated

TF genes were superinduced by CHX alone, and as discussed

by O’Connell et al. (2003), this has the potential to significantly

mask the detection of MYC2 effects on downstream target

genes. In addition, plant bHLH TFs are known to heterodimerize

with either other bHLH- or MYB-type TFs prior to binding to

target promoters. This might activate or repress transcription via

the recruitment of histone acetyltransferase or histone deacety-

lase complexes to the target promoters. Therefore, the possibility

exists that the synthesis and/or activity of a putative interacting

protein or the recruitment of coactivator or corepressor com-

plexes to MYC2 target promoters might also be influenced by

CHX. Future experiments using chromatin immunoprecipitation

followed by probing of genomic microarrays (ChIP-chip) (Lee

et al., 2007) and independent validation should be useful for the

large-scale identification of direct MYC2 targets.

In conclusion, our results reveal a number of novel functions for

MYC2 in coordinating the responses in the JA signal transduc-

tion pathway. Future work on MYC2- and JA-regulated TFs could

reveal additional information that might help us better under-

stand the regulation of this important plant hormone signaling

pathway as well as its interaction with other hormonal and

developmental signaling pathways.

METHODS

Plant Growth Conditions, Chemical Treatments, and

Pathogen Inoculations

Plant growth conditions and MeJA treatments (0.1 mM) were described

previously (Schenk et al., 2000; Campbell et al., 2003; Anderson et al.,

2004). All treatments were performed on soil-grown 4- to 5-week-old

plants, unless stated otherwise. Plants were sprayed with a 50 mM methyl

viologen (Sigma-Aldrich) solution (15 mL of solution was evenly sprayed

over 90 plants). For CHX treatments, the aboveground tissues of 4-week-

old soil-grown plants were submerged for 6 h in water containing 100 mM

CHX in large tissue culture containers. When CHX was combined with

MeJA (0.5 mM), the submerged plants were pretreated for 30 min with

CHX before the addition of MeJA.

Arabidopsis Lines and Construction of Transgenic Lines

The following Arabidopsis thaliana lines have been described elsewhere:

jin1-9 and jin1-10 (Anderson et al., 2004) and max1-1 (N9564). Seeds for

the mutant/T-DNA insertion lines were obtained from the ABRC or the

Nottingham Arabidopsis Stock Centre. The location of the T-DNA inser-

tion in the different TF genes was verified using a nested PCR approach

(Alonso et al., 2003), and homozygous plants were used in all subsequent

experiments. The mutant lines generated this way are as follows: erf2

(SALK_136141), erf6 (SALK_087356), erf11 (SALK_516053), wrky26 (SALK_

563386), wrky33 (SALK_006603), myb51 (SALK_059771), myb109 (SALK_

068392), At1g33760 (SALK_569820), and zat10 (SALK_054092).

Complementation of the jin1-9 mutant background was done as fol-

lows. The coding region of MYC2 (without the stop codon) was amplified

from genomic DNA and ligated in pENTR/D-TOPO (Invitrogen). After

sequence verification of correct amplification, the MYC2 cDNA was re-

combined into the binary vector pCTAPi (Rohila et al., 2004) using the

Gateway system (Invitrogen). Subsequent sequencing verified the correct

in-frame cloning of MYC2 fused to the CTAPi tandem affinity tag under

the control of the enhanced cauliflower mosaic virus 35S promoter. The

construct was introduced into the jin1-9 mutant background. Segregation

analysis for BASTAresistanceonT1and T2 lines allowed for theselection of

homozygous jin1-9/E35S:MYC2 lines. These lines functionally comple-

mented the jin1-9 background for inhibition of root elongation by MeJA.

Correct translation of the transgene was confirmed by protein gel blotting

with the PAP conjugate (Sigma-Aldrich) reactive against the protein A

domains of the CTAPi tag, as described before (Rivas et al., 2002).

Microarray Experiments and Data Analysis

The experimental factors of the microarray experiment were genotype

(Col-0 versus jin1-9) and treatment (6 h of 0.1 mM MeJA versus mock

controls), and for each genotype–treatment combination, three indepen-

dent biological replicates were set up. In total, these yielded 12 samples

(see Supplemental Methods online for more details). Each biological

replicate (sample) consisted of the pooled material of 30 individual

4-week-old soil-grown plants from one tray (Col-0 and jin1-9 were grown

together in a randomized design per tray). Biological replicates (trays)

were grown at different locations in the plant growth chamber and treated

separately. For details of RNA processing, ATH1 GeneChip hybridization,

and raw data collection, please see the Supplemental Methods online. All

data analysis was done using the GeneSpring software package (version

7.2; Silicon Genetics). The probe-level intensities from the CEL files were

normalized and summarized with the Robust Multi-Chip Average algo-

rithm. The resulting expression measures were then normalized per gene

to the median over the different chips. Because of the two-factor design

of the experiment, the normalized expression values were analyzed by

two-way ANOVA to determine whether either factor (genotype or treat-

ment) had a significant effect on the expression level of a certain gene.

The resulting P values (P < 0.05) were then subjected to multiple testing

correction. This resulted in the substantial reduction of significant P

values for the factor genotype, being indicative of the fact that in this

experiment, the factor treatment had an overall greater effect on gene

expression levels than the factor genotype. However, as we are primar-

ily interested in the effect of genotype on gene expression levels, we


experimentally confirmed the expression of differentially expressed

genes discussed in the text by Q-RT-PCR of the RNA samples used in

the microarray experiment and of RNA samples from an independent time

course experiment (see Results).

The lists of differentially expressed genes screened for significantly

enriched Gene Ontology terms using DAVID (Dennis et al., 2003) are

available in Supplemental Table 1 online.

Q-RT-PCR

Q-RT-PCR experiments were done as described elsewhere (McGrath

et al., 2005). The sequences of the primer pairs have been published

(Anderson et al., 2004; Czechowski et al., 2004; McGrath et al., 2005) or

can be found in Supplemental Table 3 online.

DNA Binding Assays

A 1000-bp fragment of the MYC2 coding sequence encompassing

codons 285 to 623 was amplified from genomic DNA and cloned into

the NheI-BamHI–digested pTacLCELD6�His (Xue, 2005). The resulting

construct encodes the last 338 amino acids of MYC2 (including the bHLH

region) in-frame with the reporter protein CelD and a 6xHis tag. Correct

amplification and cloning were verified by DNA sequencing. Determina-

tion of the consensus sequence of the MYC2 DNA binding motif and the

relative binding affinity of these sites was done according to Xue (2005).

Insect Feeding Experiments

All experiments were performed on 5-week-old Arabidopsis plants.

Plants were grown individually and in a completely randomized manner

in soil in a large tissue culture container, and five neonate larvae (Heli-

coverpa armigera) were placed on each plant. The containers were sealed

off with Miracloth to confine the larvae to the plant. After 6 d of feeding, the

larval weight was determined on a precision balance.

Root Growth Inhibition Assays

Surface-sterilized Arabidopsis seeds were plated on half-strength Gam-

borg’s B-5 basal medium or half-strength Murashige and Skoog medium

(supplied with 5% sucrose and 0.7% Bacto Agar, pH 6.0). Media were

supplemented with different concentrations of 5MT (Sigma-Aldrich;

solubilized in 0.1 M NaOH), MeJA (Sigma-Aldrich; solubilized in absolute

ethanol), naringenin (Sigma-Aldrich; solubilized in absolute ethanol), or

TIBA (Sigma-Aldrich; solubilized in methanol). Plates were incubated

under continuous light at 228C, and seedlings were monitored between 7

and 10 d for root growth. Root lengths were measured using the ImageJ

freeware package (http://rsb.info.nih.gov/ij/).

Measurements of Trp and Trp-Derived Metabolites

For soluble Trp, samples of 5-week-old Arabidopsis plants were frozen in

liquid nitrogen and crushed with mortar and pestle. Approximately 100

mg of the crushed material was extracted at 48C overnight in 20%

methanol. Extracts were derivatized with the AccQ-Fluor reagent kit

(Waters) and analyzed on an Acquity Ultra Performance liquid chromato-

graph (Waters).

For IGs and IAA, samples of 5-week-old Arabidopsis plants were

prepared and analyzed as described before (Sarwar and Kirkegaard,

1998; Symons and Reid, 2003).

Microarray Data Deposition

Affymetrix data have been deposited in the ArrayExpress (http://www.

ebi.ac.uk/arrayexpress/) public repository under experiment number

E-MEXP-883.

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned

in this article are as follows: MYC2 (At1g32640); ERF2 (At5g47220);

ERF6 (At4g17490); ERF11 (At1g28370); ERF4 (At3g15210), ERF13

(At2g44840); ERF14 (At1g04370); ERF8 (At1g53170); ERF9 (At5g44210);

ERF1 (At3g23240); WRKY26 (At5g07100), WRKY33 (At2g38470); MYB51

(At1g18570); MYB109 (At3g55730), ZAT10 (At1g27730); GST6

(At1g02930); CAD (At4g34230); ERF5 (At5g47230); IGPS (At2g04400);

CYP83B1/ATR4/RED1/SUR2 (At4g31500); TSA1 (At3g54640); PAD3

(At3g26830); TSB2 (At4g27070); ST5a (At1g74100); ACS6 (At4g11280);

PDF1.2 (At5g44420); HEL/PR4 (At3g04720); CHI/PR3 (At3g12500); ADC2/

SPE2 (At4g34710); VSP1 (At5g24780); MYB75/PAP1 (At1g56650);

TT19/GST12 (At5g17220); PAL1 (At2g37040); EGL3 (At1g63650); ESP

(At1g54040); ILR1 (At3g02875); MYB34/ATR1 (At5g60890); DHAR

(At1g19570); AOC4 (At1g13280); TDR1 (At3g23230); VSP2 (At5g24770),

UGT79B1 (At5g54060); APR3 (At4g21990); APS3 (At4g14680); MDHAR

(At3g09940); TAT3 (At2g24850); AACT (At5g61160); At1g33760;

At4g08870; At1g66100; At1g06160; and At3g28740.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Q-RT-PCR Expression Analysis of Selected

MYC2-Regulated Genes in jin1-9 versus Col-0 after MeJA (0.1 mM) or

Mock Treatment.

Supplemental Table 1. List of Arabidopsis Genes That Are Significantly

Affected in Their Expression by Genotype (Col-0 versus jin1-9), Treat-

ment (mock versus 0.1 mM MeJA), or Genotype–Treatment Interaction.

Supplemental Table 2. List of Arabidopsis Genes with a Strong

MYC2 Binding Site in the 3000-bp Upstream Region.

Supplemental Table 3. Sequences of the Primer Pairs Used for

Q-RT-PCR.

Supplemental Methods. MIAME-Compliant Description of Micro-

array Experiments.

ACKNOWLEDGMENTS

B.D. was the recipient of a Commonwealth Scientific and Industrial

Research Organization postdoctoral fellowship. Stephen Wilcox and his

team at the Australian Genome Research Facility in Melbourne are

gratefully acknowledged for their excellent service on the Affymetrix

experiments. We thank Andrew Fletcher for analyzing the Trp samples,

Judith Bender for providing the atr2D seeds and for useful advice on the

5MT assays, Michael Fromm for providing the CTAPi plasmid, Christine

Beveridge for the max1 mutant seeds, Sharon Downes and Tracey Parker

for providing the Helicoverpa larvae, the ABRC and the Nottingham

Arabidopsis Stock Centre for the seeds of T-DNA insertion lines, and

Anca Rusu, Christina Ehlert, Christine Bakker, Carol Kistler, Susan Batley,

Linda Krempl, and Brendan Kidd for technical assistance. We also thank

Rosanne Casu and Peter Baker for advice on GeneChip data analyses

and data presentation, Iain Wilson for critical manuscript reading, and

anonymous reviewers for critical comments on the manuscript.

Received October 10, 2006; revised May 20, 2007; accepted June 9,

2007; published July 6, 2007.

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NOTE ADDED IN PROOF

Nafisi et al. (2007) recently showed that CYP71A13 catalyzes the

conversion of indole-3-acetaldoxime in camalexin synthesis. MYC2

negatively regulates CYP71A13 (see Supplemental Table 1 online),

providing additional evidence that MYC2 is a negative regulator of JA-

dependent camalexin synthesis in Arabidopsis.

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Halkier, B.A., and Glazebrook, J. (2007). Arabidopsis cytochrome

P450 monooxygenase 71A13 catalyzes the conversion of indole-3-

acetaldoxime in camalexin synthesis. Plant Cell 19: 2039–2052.


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